Quick Denature Plasmid Sequencing Kit

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Thu May 4 15:53:49 EST 1995


In article <3o7rfq$8ek at ousrvr.oulu.fi>, jkortes at paju.oulu.fi (Jyrki
Valkama) wrote:

> Zimmermann at EMBL-Heidelberg.de wrote:
> 
> There was an interesting protocol in Biotechniques using concentrated 
> Tris for neutralization instead of HCl. It should be much more tolerant to 
> pipetting errors. (Kadokami Y, Deike CA & Lewis RV (1995)
> Biotechniques vol 18:1 pages 40-41) 

I substituted the Tris in the Rxn buffer with HEPES which is a much better
buffer at pH 7.5 .  In addition I took out the NaCl from the buffer since
one adds more than enough with the NaOH and HCl.  The success rate with
this buffer is close to 100% in my hands, the only problem being that HEPES
moves as a front on the gels (somewhere around 30 bases) so that it can be
difficult to read very close to the primers.

Zophonias
_________________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_________________________________________________________________________



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